Achievement of long term gene expression via adenoviral vector-mediated delivery of CRISPR/Cas9 for in vivo editing and gene knock-in

Background & Aim Despite its many utilities, adenoviral vectors have been of limited value for applications which require long term gene expression, such as inherited serum deficiency states. A range of technical methods have been proposed to realize long term gene expression from adenoviral vectors (Ad), however, these techniques have not gained wide employ. Nonetheless, Ads possess unique functional capacities that have qualified them to address the stringent requirements of in vivo gene therapy applications. Based on such utilities, we have exploited the emerging technology of CRISPR/Cas9-based gene editing to accomplish functional gene knock-in as a means to achieve long term corrective expression via adenoviral vectors. Methods, Results & Conclusion This technical goal is made possible by the unique ability of adenovirus to accomplish efficient and selective in vivo gene transfer. Whereas CRISPR/Cas9 technology has been shown to be compatible with adenoviral vectors, our study here is unique in exploiting Ads’ key vector capacities to achieve efficient CRISPR/Cas9 in vivo gene editing for functional knock-in of a corrective gene . To date, we have edited the ROSA26 locus in murine liver cells by inducing mutations resultant from non-homologous end joining (NHEJ) as well as successfully generating targeted insertions of a 2 kb reporter cassette of the GFP. Secondly we have shown in vivo utility of both NHEJ activity and obtained preliminary data on rates of targeted insertion of our reporter cassette. Targeted integration was achieved through co-injection of a vector containing GFP reporter cassette, flanked by ROSA26 specific sequences, and a vector encoding a ROSA26 specific guideRNA and the Cas9 endonuclease. Lastly, we tracked serum levels of episomal and integrating expression in vivo using a donor vector isogenic to our GFP reporter donor and determined integration affords extended gene expression. Serum correction models of Alpha1-antitrypsin deficiency and FIX deficiency hemophilia are being studied. By conferring upon adenovirus the ability to achieve long term gene expression, we have dramatically expanded the range of its potential applications. Our new platform technology thus represents a novel paradigm shift for vector design with true field-wide ramifications.